Methods of labeling polynucleotide with dibenzorhodamine dyes

ABSTRACT

Dibenzorhodamine compounds having the structure 
     
       
         
         
             
             
         
       
     
     are disclosed, including nitrogen- and aryl-substituted forms thereof. In addition, two intermediates useful for synthesizing such compounds are disclosed, a first intermediate having the structure 
     
       
         
         
             
             
         
       
     
     including nitrogen- and aryl-substituted forms thereof, and a second intermediate having the structure 
     
       
         
         
             
             
         
       
     
     including nitrogen- and aryl-substituted forms thereof, wherein substituents at positions C-14 to C18 taken separately are selected from the group consisting of hydrogen, chlorine, fluorine, lower alkyl, carboxylic acid, sulfonic acid, —CH 2 OH, alkoxy, phenoxy, linking group, and substituted forms thereof. The invention further includes energy transfer dyes comprising the dibenzorhodamine compounds, nucleosides labeled with the dibenzorhodamine compounds, and nucleic acid analysis methods employing the dibenzorhodamine compounds.

1. CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 11/466,085,filed Aug. 21, 2006, which is a continuation of application Ser. No.11/177,233, filed Jul. 7, 2005, which is a continuation of applicationSer. No. 10/441,950, filed May 20, 2003, which is a continuation ofapplication Ser. No. 09/969,430 filed Oct. 2, 2001 which is a divisionof application Ser. No. 09/784,943, filed Feb. 14, 2001, which is acontinuation of application Ser. No. 09/556,040, filed Apr. 20, 2000,now U.S. Pat. No. 6,221,606, which is a division of application Ser. No.09/199,402, filed Nov. 24, 1998, now U.S. Pat. No. 6,111,116, which is adivision of application Ser. No. 08/978,775, filed Nov. 25, 1997, nowU.S. Pat. No. 5,936,087, which are all incorporated herein by reference.

2. FIELD OF THE INVENTION

This invention relates generally to fluorescent dye compounds. Morespecifically, this invention relates to modified rhodamine dyes usefulas fluorescent labeling reagents.

3. BACKGROUND

The non-radioactive detection of biological analytes utilizingfluorescent labels is an important technology in modern molecularbiology. By eliminating the need for radioactive labels, safety isenhanced and the environmental impact and costs associated with reagentdisposal is greatly reduced. Examples of methods utilizing suchnon-radioactive fluorescent detection include 4-color automated DNAsequencing, oligonucleotide hybridization methods, detection ofpolymerase-chain-reaction products, immunoassays, and the like.

In many applications it is advantageous to employ multiple spectrallydistinguishable fluorescent labels in order to achieve independentdetection of a plurality of spatially overlapping analytes, e.g.,single-tube multiplex DNA probe assays and 4-color automated DNAsequencing methods. In the case of multiplex DNA probe assays, byemploying spectrally distinguishable fluorescent labels, the number ofreaction tubes may be reduced thereby simplifying experimental protocolsand facilitating the production of application-specific reagent kits. Inthe case of 4-color automated DNA sequencing, multicolor fluorescentlabeling allows for the analysis of multiple bases in a single lanethereby increasing throughput over single-color methods and reducinguncertainties associated with inter-lane electrophoretic mobilityvariations.

Assembling a set of multiple spectrally distinguishable fluorescentlabels is problematic. Multi-color fluorescent detection imposes atleast six severe constraints on the selection of dye labels,particularly for applications requiring a single excitation lightsource, an electrophoretic separation, and/or treatment with enzymes,e.g., automated DNA sequencing. First, it is difficult to find a set ofstructurally similar dyes whose emission spectra are spectrallyresolved, since the typical emission band half-width for organicfluorescent dyes is about 40-80 nanometers (nm). Second, even if dyeswith non-overlapping emission spectra are identified, the set may stillnot be suitable if the respective fluorescent quantum efficiencies aretoo low. Third, when several fluorescent dyes are used concurrently,simultaneous excitation becomes difficult because the absorption bandsof the dyes are usually widely separated. Fourth, the charge, molecularsize, and conformation of the dyes must not adversely affect theelectrophoretic mobilities of the analyte. Fifth, the fluorescent dyesmust be compatible with the chemistry used to create or manipulate theanalyte, e.g., DNA synthesis solvents and reagents, buffers, polymeraseenzymes, ligase enzymes, and the like. Sixth, the dye must havesufficient photostability to withstand laser excitation.

Currently available multiplex dye sets suitable in 4-color automated DNAsequencing applications require blue or blue-green laser light toadequately excite fluorescence emissions from all of the dyes making upthe set, e.g., argon-ion lasers. Use of Blue or blue-green lasers incommercial automated DNA sequencing systems is disadvantageous becauseof the high cost and limited lifetime of such lasers.

4. SUMMARY

The present invention is directed towards our discovery of a class ofdibenzorhodamine dye compounds suitable for the creation of sets ofspectrally-resolvable fluorescent labels useful for multi-colorfluorescent detection. The subject dye compounds are particularly wellsuited for use in automated 4-color DNA sequencing systems using anexcitation light source having a wavelength greater than about 630 nm,e.g., a helium-neon gas laser or a solid state diode laser.

In a first aspect, the invention comprises dibenzorhodamine dyecompounds having the structure

including nitrogen- and aryl-substituted forms thereof.

In a second aspect, the invention comprises intermediates useful for thesynthesis of dibenzorhodamine compounds having the structure

including nitrogen- and aryl-substituted forms thereof.

In a third aspect, the invention comprises intermediates useful for thesynthesis of dibenzorhodamine compounds having the structure

including nitrogen- and aryl-substituted forms thereof, wherein R₁ takentogether with the C-12-bonded nitrogen and the C-12 and C-13 carbonsforms a first ring structure having from 4 to 7 members; and/or R₁ takentogether with the C-12-bonded nitrogen and the C-11 and C-12 carbonsforms a second ring structure having from 5 to 7 members.

In a fourth aspect, the invention includes energy transfer dye compoundscomprising a donor dye, an acceptor dye, and a linker linking the donorand acceptor dyes. The donor dye is capable of absorbing light at afirst wavelength and emitting excitation energy in response, and theacceptor dye is capable of absorbing the excitation energy emitted bythe donor dye and fluorescing at a second wavelength in response. Thelinker serves to facilitate the efficient transfer of energy between thedonor dye and the acceptor dye. According to the present invention, atleast one of the donor and acceptor dyes is a dibenzorhodamine dyehaving the structure set forth above.

In a fifth aspect, the present invention includes labelednucleoside/tides having the structure

NUC-D

wherein NUC is a nucleoside/tide or nucleoside/tide analog and D is adibenzorhodamine dye compound having the structure set forth above.According to the invention, NUC and D are connected by a linkage whereinthe linkage is attached to D at one of the substituent positions.Furthermore, if NUC comprises a purine base, the linkage is attached tothe 8-position of the purine, if NUC comprises a 7-deazapurine base, thelinkage is attached to the 7-position of the 7-deazapurine, and if NUCcomprises a pyrimidine base, the linkage is attached to the 5-positionof the pyrimidine.

In a sixth aspect, the invention includes polynucleotide analysismethods comprising the steps of forming a set of labeled polynucleotidefragments labeled with a dibenzorhodamine dye having the structure setforth above, subjecting the labeled polynucleotide fragments to asize-dependent separation process, e.g., electrophoresis, and detectingthe labeled polynucleotide fragments subsequent to the separationprocess.

Various aspects of the above-described invention achieve one or more ofthe following important advantages over known fluorescent dye compoundsuseful for multiplex fluorescent detection: (1) the subject dyecompounds may be efficiently excited by a low-cost red laser usingwavelengths at or above 630 nm; (2) the emission spectra of the subjectdye compounds can be modulated by minor variations in the type andlocation of nitrogen and/or aryl-substituents, allowing for the creationof dye sets having similar absorption characteristics yet spectrallyresolvable fluorescence emission spectra; (3) the subject dye compoundsmay be easily attached to nucleosides/tides or polynucleotides withoutcompromising their favorable fluorescence properties; (4) the subjectdye compounds have narrow emission bandwidths, i.e., the emissionbandwidth has a full-width at half the maximum emission intensity ofbelow about 50 nm; (5) the subject dye compounds are highly soluble inbuffered aqueous solution while retaining a high quantum yield; (6) thesubject dye compounds are relatively photostable; and (7) the subjectdye compounds have relatively large extinction coefficients, i.e.,greater than about 50,000.

These and other features and advantages of the present invention willbecome better understood with reference to the following description,figures, and appended claims.

5. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1-3 show exemplary synthetic pathways for the synthesis of the1-amino-3-hydroxynapthalene intermediates of the invention.

FIG. 4 shows a generalized synthetic pathway for the synthesis of thedibenzorhodamine dye compounds of the invention.

FIGS. 5 and 6 show exemplary synthetic pathways for the synthesis of thedibenzorhodamine dye compounds of the invention.

FIG. 7 shows the structures of several exemplary dibenzorhodamine dyecompounds of the invention.

6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference will now be made in detail to the preferred embodiments of theinvention, examples of which are illustrated in the accompanyingdrawings. While the invention will be described in conjunction with thepreferred embodiments, it will be understood that they are not intendedto limit the invention to those embodiments. On the contrary, theinvention is intended to cover all alternatives, modifications, andequivalents, which may be included within the invention as defined bythe appended claims.

Generally, the present invention comprises a novel class ofdibenzorhodamine dye compounds useful as fluorescent labels, methods andintermediates for synthesis of such dyes, reagents employing such dyes,and methods utilizing such dyes and reagents in the area of analyticalbiotechnology. The compounds of the present invention find particularapplication in the area of fluorescent nucleic acid analysis, e.g.,automated DNA sequencing and fragment analysis, detection of probehybridization in hybridization arrays, detection of nucleic acidamplification products, and the like.

1.1. DEFINITIONS

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings:

“Spectral resolution” in reference to a set of dyes means that thefluorescent emission bands of the dyes are sufficiently distinct, i.e.,sufficiently non-overlapping, that reagents to which the respective dyesare attached, e.g. polynucleotides, can be distinguished on the basis ofa fluorescent signal generated by the respective dyes using standardphotodetection systems, e.g. employing a system of band pass filters andphotomultiplier tubes, charged-coupled devices and spectrographs, or thelike, as exemplified by the systems described in U.S. Pat. Nos.4,230,558, 4,811,218, or in Wheeless et al, pgs. 21-76, in FlowCytometry: Instrumentation and Data Analysis (Academic Press, New York,1985).

“Electron-rich heterocycle” means cyclic compounds in which one or morering atoms are not carbon, i.e., are hetero atoms, and the heteroatomshave unpaired electrons which contribute to a 6-π electronic system.Exemplary electron-rich heterocycles include but are not limited topyrrole, indole, furan, benzofuran, thiophene, benzothiophene and otherlike structures.

“Linking group” means a moiety capable of reacting with a “complementaryfunctionality” attached to a reagent or member of an energy transfer dyepair, such reaction forming a “linkage” connecting the dye to thereagent or member of the energy transfer dye pair. Preferred linkinggroups include isothiocyanate, sulfonyl chloride, 4,6-dichlorotriazinyl,succinimidyl ester, or other active carboxylate whenever thecomplementary functionality is amine. Preferably the linking group ismaleimide, halo acetyl, or iodoacetamide whenever the complementaryfunctionality is sulfhydryl. See R. Haugland, Molecular Probes Handbookof Fluorescent Probes and Research Chemicals, Molecular probes, Inc.(1992). In a particularly preferred embodiment, the linking group is aN-hydroxysuccinimidyl (NHS) ester and the complementary functionality isan amine, where to form the NHS ester, a dye of the invention includinga carboxylate linking group is reacted with dicyclohexylcarbodiimide andN-hydroxysuccinimide.

“Substituted” as used herein refers to a molecule wherein one or morehydrogen atoms are replaced with one or more non-hydrogen atoms,functional groups or moieties. For example, an unsubstituted nitrogen is—NH₂, while a substituted nitrogen is —NHCH₃. Exemplary substituentsinclude but are not limited to halo, e.g., fluorine and chlorine, loweralkyl, lower alkene, lower alkyne, sulfate, sulfonate, sulfone, amino,ammonium, amido, nitrile, lower alkoxy, phenoxy, aromatic, phenyl,polycyclic aromatic, electron-rich heterocycle water-solubilizing group,and linking group.

“Polycyclic aromatic” means aromatic hydrocarbons having multiple ringstructures including biaryls and condensed benzenoid hydrocarbons. Thebiaryls are benzenoid compounds where two or more rings are linkedtogether by a single bond. The parent system of this class is biphenyl.The condensed benzenoid compounds are characterized by two or morebenzene rings fused together at ortho positions in such a way that eachpair of rings shares two carbons. The simplest members of this group arenapthalene, with two rings, and anthracene and phenanthrene, each withthree rings.

“Lower alkyl” denotes straight-chain and branched hydrocarbon moietiescontaining from 1 to 8 carbon atoms, e.g., methyl, ethyl, propyl,isopropyl, tert-butyl, isobutyl, sec-butyl, neopentyl, tert-pentyl, andthe like.

“Lower alkene” denotes a hydrocarbon containing from 1 to 8 carbon atomswherein one or more of the carbon-carbon bonds are double bonds.

“Lower alkyne” denotes a hydrocarbon containing from 1 to 8 carbon atomswherein one or more of the carbons are bonded with a triple bond.

“Nucleoside” refers to a compound consisting of a purine, deazapurine,or pyrimidine nucleoside base, e.g., adenine, guanine, cytosine, uracil,thymine, deazaadenine, deazaguanosine, and the like, linked to a pentoseat the 1′ position. When the nucleoside base is purine or 7-deazapurine,the sugar moiety is attached at the 9-position of the purine ordeazapurine, and when the nucleoside base is pyrimidine, the sugarmoiety is attached at the 1-position of the pyrimidine, e.g., Kornbergand Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992). Theterm “nucleotide” as used herein refers to a phosphate ester of anucleoside, e.g., triphosphate esters, wherein the most common site ofesterification is the hydroxyl group attached to the C-5 position of thepentose. The term “nucleoside/tide” as used herein refers to a set ofcompounds including both nucleosides and nucleotides. “Analogs” inreference to nucleosides/tides include synthetic analogs having modifiedbase moieties, modified sugar moieties and/or modified phosphatemoieties, e.g. described generally by Scheit, Nucleotide Analogs (JohnWiley, New York, 1980). Phosphate analogs comprise analogs of phosphatewherein the phosphorous atom is in the +5 oxidation state and one ormore of the oxygen atoms is replaced with a non-oxygen moiety. Exemplaryanalogs include but are not limited to phosphorothioate,phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,phosphoroanilothioate, phosphoranilidate, phosphoramidate,boronophosphates, including associated counterions, e.g., H⁺, NH₄ ⁺,Na⁺, if such counterions are present. Exemplary base analogs include butare not limited to 2,6-diaminopurine, hypoxanthine, pseudouridine,C-5-propyne, isocytosine, isoguanine, 2-thiopyrimidine, and other likeanalogs. Exemplary sugar analogs include but are not limited to 2′- or3′-modifications where the 2′- or 3′-position is hydrogen, hydroxy,alkoxy, e.g., methoxy, ethoxy, allyloxy, isopropoxy, butoxy, isobutoxyand phenoxy, amino or alkylamino, fluoro, chloro and bromo. The term“labeled nucleoside/tide” refers to nucleosides/tides which arecovalently attached to the dye compounds of Formula I through a linkage.

“Water solubilizing group” means a substituent which increases thesolubility of the compounds of the invention in aqueous solution.Exemplary water-solubilizing groups include but are not limited toquaternary amine, sulfate, sulfonate, carboxylate, phosphate, polyether,polyhydroxyl, and boronate.

“Polynucleotide” or “oligonucleotide” means polymers of naturalnucleotide monomers or analogs thereof, including double and singlestranded deoxyribonucleotides, ribonucleotides, α-anomeric formsthereof, and the like. Usually the nucleoside monomers are linked byphosphodiester linkages, where as used herein, the term “phosphodiesterlinkage” refers to phosphodiester bonds or bonds including phosphateanalogs thereof, including associated counterions, e.g., H⁺, NH₄ ⁺, Na⁺,if such counterions are present. Polynucleotides typically range in sizefrom a few monomeric units, e.g. 5-40, to several thousands of monomericunits. Whenever a polynucleotide is represented by a sequence ofletters, such as “ATGCCTG,” it will be understood that the nucleotidesare in 5′->3′ order from left to right and that “A” denotesdeoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine,and “T” denotes deoxythymidine, unless otherwise noted.

“Rhodamine dye” refers to dyes including the general polycyclicstructure

including any and all substituted versions thereof.

1.2. 1-AMINO-3-HYDROXYNAPTHALENE INTERMEDIATES 1.2.1. Structure

In a first aspect, the present invention comprises a novel class of1-amino-3-hydroxynapthalene compounds useful as intermediates in thesynthesis of dibenzorhodamine dyes. These compounds have the generalstructure shown in Formula I immediately below, including substitutedforms thereof, where R₁ taken together with the C-12-bonded nitrogen andthe C-12 and C-13 carbons forms a first ring structure having from 4 to7 members; and/or R₁ taken together with the C-12-bonded nitrogen andthe C-11 and C-12 carbons forms a second ring structure having from 5 to7 members. (Note that all molecular structures provided herein areintended to encompass not only the exact electronic structurespresented, but also include all resonant structures, protonation statesand associated counterions thereof.)

In the compound of Formula I, preferably the first ring structure hasfive members. More preferably, the five membered first ring structureincludes one gem disubstituted carbon, e.g., wherein the gemsubstituents are lower alkyl, e.g., methyl. In an alternative preferredembodiment, the five membered ring is substituted with linking group orwater-solubilizing group.

In another preferred embodiment of the intermediate of Formula I, thesecond ring structure has six members. More preferably, the six-memberedsecond ring structure includes one gem disubstituted carbon, e.g.,wherein the gem substituents are lower alkyl, e.g., methyl. In analternative preferred embodiment, the five membered ring is substitutedwith linking group.

Preferably, the compound of Formula I includes one or more nitrogensubstituents. Exemplary nitrogen substituents include but are notlimited to lower alkyl, lower alkene, lower alkyne, phenyl, aromatic,electron-rich heterocycle, polycyclic aromatic, water-solubilizinggroup, and linking group, including substituted forms thereof. In aparticularly preferred embodiment, the nitrogen substituents are loweralkyl and/or phenyl, including substituted forms thereof. Morepreferably, the nitrogen substituents are substituted lower alkyl orsubstituted phenyl, wherein the substituent is linking group, orwater-solubilizing group.

In an additional preferred embodiment, one or more of carbons atpositions C-8 to C-11 are substituted. Exemplary substituents includebut are not limited to fluorine, chlorine, lower alkyl, lower alkene,lower alkyne, sulfate, sulfonate, sulfone, sulfonamide, sulfoxide,amino, ammonium, amido, nitrile, lower alkoxy, phenoxy, aromatic,phenyl, polycyclic aromatic, electron-rich heterocycle,water-solubilizing group, and linking group, including substituted formsthereof. Preferably, one or more of the substituents is sulfonate.

Several representative 1-amino-3-hydroxynapthalene compounds of theinvention are shown in FIGS. 1-3, i.e., compounds 4, 9, 15, 17, 22, 27and 29.

1.2.2. Synthetic Methods

Several synthetic methods are available for the synthesis of the1-amino-3-hydroxynapthalene compounds described above, different methodsbeing preferred depending on the nature of the ring structure and thenitrogen substituents of the particular compound to be synthesized.

A first preferred synthesis method suitable for the synthesis of1-substituted-amino-3-hydroxynapthalene compounds, e.g.,1-diethylamino-3-hydroxynapthalene 4, is shown in FIG. 1. In this firstmethod, a 3-methoxy-1-hydroxy napthalene 1 is reacted with drytriethylamine and trifluoromethanesulfonic anhydride to form a crude3-methoxynapthalene-1-triflate 2. The triflate 2 is then reacted with anamine, e.g., a secondary amine, e.g., diethylamine, using palladiumcatalyzed triflate/amine coupling to form the substituted amine compound3. Compound 3 is then deprotected using a boron tribromide deprotectionprocedure to produce the 1-amino-3-hydroxynapthalene product, e.g.,1-diethylamino-3-hydroxynapthalene 4. An example of this synthesis isprovided in Example 1 below.

A second preferred synthesis method suitable for the synthesis ofbenzoindoline compounds, e.g.,N-phenyl-3,3-dimethyl-4-hydroxy-benzoindoline 9, is also shown inFIG. 1. In this method, the 3-methoxynapthalene-1-triflate 2 isderivatized with a primary amine, e.g., aniline, using a palladiumcatalyzed triflate coupling reaction to give a secondary amine, e.g.,1-anilino-3-methoxynapthalene 5. The secondary amine 5 is acetylatedusing an acid chloride, e.g., an haloacetylchloride, to give adisubstituted amide, e.g., 1-amido-3-methoxynapthalene 6. The tertiaryamide 6 is cyclized using a Lewis-acid-catalyzed Friedel-Craftscyclization procedure to give compound 7, e.g., using AlCl₃. Compound 7is than reduced, e.g., using LAH, to give compound 8. Subsequent methoxygroup deprotection by a boron tribromide deprotection procedure givesthe benzoindoline, e.g., N-phenyl-3,3-dimethyl-4-hydroxy-benzoindoline9. An example of this synthesis is provided in Example 2 below.

A third preferred synthesis method suitable for the synthesis ofN-substituted-5-hydroxy-(tetrahydro)benzoquinoline compounds, e.g.,N-methyl-5-hydroxy-(tetrahydro)benzoquinoline 15, is shown in FIG. 2. Inthis method, compound 10 is synthesized from methoxy-napthaldehyde bycondensation with malonic acid using a piperidine catalyst in pyridine.Compound 10 is then reduced with hydrogen, followed by LAH reduction,and reacted with trifluoromethanesulfonic anhydride to give the triflate11. The triflate 11 is reacted with NaN₃ to give compound 12. Compound12 is complexed with a Lewis acid, e.g., AlCl₃, and refluxed yieldingthe cyclized benzoquinoline derivative 13. Next, a nitrogen substituentis added, e.g., the nitrogen is alkylated using a conventionalalkylation procedure, e.g., the benzoquinoline derivative 13 is reactedwith n-butyl lithium and an alkylating agent, e.g., MeI to give compound14 or propane sultone to give compound 16. The methoxy group is thenremoved by a boron tribromide procedure giving a N-alkylbenzoquinolinederivative, e.g., compound 15 or 17. An example of this synthesis isprovided in Example 3 below.

A fourth preferred synthesis method suitable for the synthesis ofN-substituted-2,2,4-trimethyl-5-hydroxy-benzoquinoline compounds, e.g.N-methyl-2,2,4-trimethyl-5-hydroxy-(tetrahydro)benzoquinoline 22, isshown in FIG. 3. In this method, following the procedure of A. Rosowskyand E. J. Modest (J.O.C. 30 1832 1965, and references therein),1-amino-3-methoxynapthalene 18 is reacted with acetone catalyzed byiodine and then quenched with saturated Na₂S₂O₃ to give thebenzoquinoline compound 19. Compound 19 is then alkylated with analkylating agent, e.g., MeI, according to a general alkylation procedureto give compound 20. The alkylated compound 20 is reduced with H₂catalyzed by Pd/C to give a N-methyl-methoxyquinoline intermediate 21,and subsequent methoxy group deprotection by a general boron tribromideprocedure yields theN-substituted-2,2,4-trimethyl-5-hydroxy-benzoquinoline compound, e.g.,N-methyl-2,2,4-trimethyl-5-hydroxy-(tetrahydro)benzoquinoline 22. Anexample of this synthesis is provided in Example 4 below.

A fifth preferred general synthesis method suitable for the synthesis ofN-substituted-3,3-dimethyl-4-hydroxy-benzoindoline compounds, e.gN-methyl-3,3-dimethyl-4-hydroxy-benzoindoline 27, is also shown in FIG.3. In this method, a 1-amino-3-methoxynapthalene 18 is acetylated withan acid chloride, e.g., 2-bromo-2-methylpropionyl chloride, to givecompound 23. Compound 23 is cyclized by reaction with AlCl₃ to givecompound 24. Compound 24 is then reduced with LAH to give the3,3-dimethyl-4-methoxybenzoindoline 25. Compound 25 is then alkylatedwith an alkylating agent, e.g., methyl iodide, to give aN-methyl-3,3-dimethyl-4-methoxybenzoindoline, e.g., compound 26.Subsequent methoxy group deprotection by with boron tribromide givescompound 27. An example of this synthesis is provided in Example 5below.

1.3. DIBENZORHODAMINE DYE COMPOUNDS 1.3.1. Structure

In a second aspect, the present invention comprises a novel class ofdibenzorhodamine dye compounds useful as molecular labels having thegeneral structure shown in Formula II immediately below, including aryl-and nitrogen-substituted forms thereof.

In one preferred embodiment of the compound of Formula II, the compoundincludes a first bridging group which when taken together with theC-12-bonded nitrogen and the C-12 and C-13 carbons forms a first ringstructure having from 4 to 7 members, and/or a second bridging groupwhich when taken together with the C-2-bonded-nitrogen and the C-1 andC-2 carbons forms a second ring structure having from 4 to 7 members.More preferably, one or both of the first and second ring structures hasfive members. In yet a more preferred embodiment, the five membered ringstructure includes one gem disubstituted carbon, wherein the gemsubstituents are lower alkyl, e.g., methyl. In an alternative preferredembodiment, the five membered ring is substituted with linking group.

In another preferred embodiment, the compound of Formula II includes aC-7 substituent selected from the group consisting of acetylene, loweralkyl, lower alkene, cyano, phenyl, heterocyclic aromatic, electron-richheterocycle, and substituted forms thereof. In a more preferredembodiment, the C-7 substituent is a phenyl or substituted phenyl havingthe structure

wherein aryl substituents at positions C-14 to C-18 taken separately maybe selected from the group consisting of hydrogen, chlorine, fluorine,lower alkyl, carboxylic acid, sulfonic acid, —CH₂OH, alkoxy, phenoxy,linking group, and substituted forms thereof. Preferably, the phenylsubstituent at C-18 is selected from the group consisting of carboxylicacid and sulfonate, and is most preferably carboxylic acid. In anotherpreferred embodiment, substituents at positions C-14 and C-17 arechlorine. In yet another preferred embodiment, substituents at positionsC-14 to C-17 are all chlorine or all fluorine. In a particularlypreferred embodiment, substituents at one of positions C-15 and C-16 islinking group and the other is hydrogen, substituents at positions C-14and C-17 are chlorine, and a substituent at position C-18 is carboxy.

In yet another preferred embodiment of the invention, the compound ofFormula II includes one or more nitrogen substituents. Preferably, suchsubstituents are selected from the group consisting of lower alkyl,lower alkene, lower alkyne, phenyl, aromatic, electron-rich heterocycle,polycyclic aromatic, water-solubilizing group, linking group, andsubstituted forms thereof. More preferably, the nitrogen substituentsare selected from the group consisting of lower alkyl, phenyl, andsubstituted forms thereof, where exemplary substituents include linkinggroup, and water-solubilizing group.

In another preferred embodiment of this second aspect of the invention,the compound of Formula II includes a third bridging group which whentaken together with the C-12-bonded nitrogen and the C-11 and C-12carbons forms a third ring structure having from 5 to 7 members, and/ora fourth bridging group which when taken together with the C-2-bondednitrogen and the C-2 and C-3 carbons forms a fourth ring structurehaving from 5 to 7 members. Preferably, one or both of the third andfourth ring structures has six members. More preferably, the sixmembered ring structure includes one gem disubstituted carbon, whereinthe gem substituents are lower alkyl, e.g., methyl.

In another preferred embodiment of the invention, the compound ofFormula II includes aryl substituents at one or more of carbons C-1, C-3through C-6, C-8 through C-11, and C-13. Exemplary aryl substituentsinclude but are not limited to fluorine, chlorine, lower alkyl, loweralkene, lower alkyne, sulfate, sulfonate, sulfone, sulfonamide,sulfoxide, amino, ammonium, amido, nitrile, lower alkoxy, phenoxy,aromatic, phenyl, polycyclic aromatic, water-solubilizing group,electron-rich heterocycle, and linking group, including substitutedforms thereof. In a particularly preferred embodiment, at least onesubstituent is sulfonate.

Several exemplary dye compounds according to this second aspect of theinvention are shown in FIG. 7, i.e., compounds 41-47.

1.3.2. Synthetic Methods

Generally, the dibenzorhodamine dyes of the present invention aresynthesized as follows. See FIG. 4. An anhydride derivative 30, e.g., aphthalic anhydride, is mixed with 1-amino-3-methoxy intermediates 31 and32, and Lewis acid, e.g., ZnCl₂, where the R— substituents in compound30 may be the same or different, but are preferably the same. ExemplaryR-substituents include but are not limited to acetylene, lower alkyl,lower alkene, phenyl, heterocyclic aromatic, electron-rich heterocycle,and substituted forms thereof. The mixture is heated briefly untilmelting is observed. A solvent, e.g., 1,2-dichlorobenzene, is added tothe reaction mixture, and the heterogeneous mixture is heated to about130° C. to about 180° C. The crude reaction mixture is cooled andpurified by normal phase flash chromatography to yield dye compound 33.When the anhydride is part of a substituted phthalic anhydride, e.g.,compound 34, two isomers are formed. See FIG. 5. The isomers 35 and 36are separated by PTLC. The isomerically pure dyes are identified bysingle spots on normal and reverse phase TLC and by their UV/Visibleabsorption spectra and their long wavelength fluorescent excitation andemission spectra.

An alternative procedure for the synthesis of asymmetrical dye compoundsis shown in FIG. 6. In this process, an anhydride derivative, e.g.,phthalic anhydride 34, is mixed with dry nitrobenzene and heated. Themixture is cooled to room temperature and anhydrous AlCl₃ is added withstirring. Subsequently a 1-amino-3-methoxy intermediate 31 is added withstirring and the reaction is heated. The reaction is cooled andsuspended in EtOAc. The organic layer is washed with saturated NH₄Cl,brine, dried over Na₂SO₄, filtered, and the solvent removed in vacuo.The resulting ketone intermediates 37/38 are purified and separated intodistinct isomers 37 and 38 (except where substituents at C-14 and C-17are the same and substituents at C-15 and C-16 are the same) by flashchromatography or recrystallization. The methoxy group of theisomerically pure ketone intermediate 37 or 38 is removed according to ageneral boron tribromide deprotection procedure to give theamino-hydroxynapthalene ketone intermediate 39. Amino-hydroxynapthaleneketone intermediate 39 is then reacted with a 1-amino-3-methoxyintermediate 32. The reaction is cooled, giving isomerically pure andasymmetrically substituted product 40 that may be further purified byPTLC.

1.4. ENERGY TRANSFER DYES INCORPORATING THE DIBENZORHODAMINE DYES

In another aspect, the present invention comprises energy transfer dyecompounds incorporating the dibenzorhodamine dye compounds of Formula I.Generally, the energy transfer dyes of the present invention include adonor dye which absorbs light at a first wavelength and emits excitationenergy in response, an acceptor dye which is capable of absorbing theexcitation energy emitted by the donor dye and fluorescing at a secondwavelength in response, and a linker which attaches the donor dye to theacceptor dye, the linker being effective to facilitate efficient energytransfer between the donor and acceptor dyes. A through discussion ofthe structure, synthesis and use of such energy transfer dyes isprovided by Lee et al., U.S. patent application Ser. No. 08/726,462, andMathies et al., U.S. Pat. No. 5,654,419.

One linker according to the present invention for linking a donor dye toan acceptor dye in an energy transfer fluorescent dye has the generalstructure

where R₂₁ is a lower alkyl attached to the donor dye, Z₁ is either NH,sulfur or oxygen, R₂₂ is a substituent which includes an alkene, diene,alkyne, a five and six membered ring having at least one unsaturatedbond or a fused ring structure which is attached to the carbonyl carbon,and R₂₈ includes a functional group which attaches the linker to theacceptor dye.

In one embodiment of this linker, illustrated below, the linker has thegeneral structure

where R₂₁ and R₂₂ are as detailed above, Z₁ and Z₂ are eachindependently either NH, sulfur or oxygen, R₂₉ is a lower alkyl, and theterminal carbonyl group is attached to a ring structure of the acceptordye. In the variation where Z₂ is nitrogen, the C(O)R₂₂R₂₉Z₂ subunitforms an amino acid subunit. Particular examples of five or six memberedrings which may be used as R₂₂ in the linker include, but are notlimited to cyclopentene, cyclohexene, cyclopentadiene, cyclohexadiene,furan, thiofuran, pyrrole, pyrazole, isoimidazole, pyran, pyrone,benzene, pyridine, pyridazine, pyrimidine, triazine, pyrazine andoxazine. Examples of fused ring structures include, but are not limitedto indene, benzofuran, thionaphthene, indole and naphthalene. Apreferred embodiment of this linker is where R₂₁ and R₂₉ are methylene,Z₁ and Z₂ are NH, and R₂₂ is benzene, as shown below.

In another preferred embodiment of the energy-transfer-dye aspect of thepresent invention, the linker attaches to the dibenzorhodamine dyecomponent of the energy transfer dye at the C-1 or 13 positions, or,alternatively, where the C-7 substituent is phenyl or substitutedphenyl, at one of the C-15 or C-16 positions. In a particularlypreferred embodiment, both members of the energy transfer pair aredibenzorhodamine dyes, and the first member is linked through the C-1position and the second member is linked through one of the C-15 or C-16positions.

In yet another preferred embodiment of the energy-transfer-dye aspect ofthe present invention, a first member the dye pair is a dibenzorhodaminedye, and a second member of the dye pair is cyanine, phthalocyanine,squaraine, bodipy, fluorescein, or dibenzorhodamine dye having differentsubstitutions than the first member.

1.5. REAGENTS INCORPORATING THE DIBENZORHODAMINE DYES

In another aspect, the present invention comprises reagents labeled withthe dibenzorhodamine dye compounds of Formula I. Reagents of theinvention can be virtually anything to which the dyes of the inventioncan be attached. Preferably, the dye is covalently attached to thereagent. Reagents may include but are not limited to proteins,polypeptides, polysaccharides, nucleotides, nucleosides,polynucleotides, lipids, solid supports, organic and inorganic polymers,and combinations and assemblages thereof, such as chromosomes, nuclei,living cells, such as bacteria or other microorganisms, mammalian cells,tissues, and the like.

1.5.1. Nucleoside/Tide Reagents

A preferred class of labeled reagents comprise nucleoside/tides thatincorporate the dibenzorhodamine dyes of the invention. Suchnucleoside/tide reagents are particularly useful in the context oflabeling polynucleotides formed by enzymatic synthesis, e.g., nucleotidetriphosphates used in the context of PCR amplification, Sanger-typepolynucleotide sequencing, and nick-translation reactions.

Generally, the structure of the labeled nucleoside/tide reagent is

NUC-D  FORMULA III

where NUC is a nucleoside/tide or nucleoside/tide analog and D is adibenzorhodamine dye compound of Formula II.

The linkage linking the nucleoside/tide and the dye may be attached tothe dye at any one of substituent positions C-1 to C-18 or at a C-2bonded nitrogen or a C-12 bonded nitrogen. Preferably, the dye includesa phenyl or substituted phenyl substituent at the C-7 position and isattached to the nucleoside/tide at one of the C-15 or C-16 substituentpositions, the other position being a hydrogen atom.

When NUC includes a purine base, the linkage between NUC and D isattached to the N⁸-position of the purine, when NUC includes a7-deazapurine base, the linkage is attached to the N⁷-position of the7-deazapurine, and when NUC includes a pyrimidine base, the linkage isattached to the N⁵-position of the pyrimidine.

Nucleoside labeling can be accomplished using any one of a large numberof known nucleoside/tide labeling techniques employing known linkages,linking groups, and associated complementary functionalities. Generally,the linkage linking the dye and nucleoside should (i) not interfere witholigonucleotide-target hybridization, (ii) be compatible with relevantenzymes, e.g., polymerases, ligases, and the like, and (iii) notadversely affect the fluorescence properties of the dye. Exemplary baselabeling procedures suitable for use in connection with the presentinvention include the following: Gibson et al, Nucleic Acids Research,15:6455-6467 (1987); Gebeyehu et al, Nucleic Acids Research, 15:4513-4535 (1987); Haralambidis et al, Nucleic Acids Research, 15:4856-4876 (1987); Nelson et al., Nucleosides and Nucleotides, 5(3):233-241 (1986); Bergstrom, et al., JACS, 111: 374-375 (1989); and U.S.Pat. Nos. 4,855,225, 5,231,191, and 5,449,767.

Preferably, the linkages are acetylenic amido or alkenic amido linkages,the linkage between the dye and the nucleoside/tide base being formed byreacting an activated N-hydroxysuccinimide (NHS) ester of the dye withan alkynylamino- or alkenylamino-derivatized base of a nucleoside/tide.More preferably, the resulting linkage is 3-(carboxy)amino-1-propyn-1-ylhaving the structure

Alternative preferred linkages include substituted propargylethoxyamidolinkages having the structure

NUC-C≡C—CH₂OCH₂CH₂NR₃X-D

wherein X is selected from the group consisting of

where n ranges from 1 to 5,

where n ranges from 1 to 5,

R₁ is selected from the group consisting of —H, lower alkyl andprotecting group; and R₃ is selected from the group consisting of —H andlower alkyl. See Khan et al., U.S. patent application Ser. No.08/833,854 filed Apr. 10, 1997.

The synthesis of alkynylamino-derivatized nucleosides is taught by Hobbset al. in European Patent Application No. 87305844.0, and Hobbs et al.,J. Org. Chem., 54: 3420 (1989). Briefly, the alkynylamino-derivatizednucleotides are formed by placing the appropriate halodideoxynucleoside(usually 5-iodopyrimidine and 7-iodo-7-deazapurine dideoxynucleosides astaught by Hobbs et al. (cited above)) and Cu(I) in a flask, flushingwith argon to remove air, adding dry DMF, followed by addition of analkynylamine, triethyl-amine and Pd(0). The reaction mixture is stirredfor several hours, or until thin layer chromatography indicatesconsumption of the halodideoxynucleoside. When an unprotectedalkynylamine is used, the alkynylamino-nucleoside can be isolated byconcentrating the reaction mixture and chromatographing on silica gelusing an eluting solvent which contains ammonium hydroxide to neutralizethe hydrohalide generated in the coupling reaction. When a protectedalkynylamine is used, methanol/methylene chloride can be added to thereaction mixture, followed by the bicarbonate form of a strongly basicanion exchange resin. The slurry can then be stirred for about 45minutes, filtered, and the resin rinsed with additionalmethanol/methylene chloride. The combined filtrates can be concentratedand purified by flash-chromatography on silica gel using amethanol-methylene chloride gradient. The triphosphates are obtained bystandard techniques.

Particularly preferred nucleosides/tides of the present invention areshown below in Formula IV wherein

B is a nucleoside/tide base, e.g., uracil, cytosine, deazaadenine, ordeazaguanosine; W₁ and W₂ taken separately are OH or a group capable ofblocking polymerase-mediated template-directed polymerization, e.g., H,fluorine and the like; W₃ is OH, or mono-, di- or triphosphate orphosphate analog; and D is a dye compound of Formula I. In oneparticularly preferred embodiment, the nucleotides of the presentinvention are dideoxynucleotide triphosphates having the structure shownin Formula IV.1 below, including associated counterions if present.

Labeled dideoxy nucleotides such as that shown in Formula IV.1 findparticular application as chain terminating agents in Sanger-type DNAsequencing methods utilizing fluorescent detection.

In a second particularly preferred embodiment, the nucleotides of thepresent invention are deoxynucleotide triphosphates having the structureshown in Formula IV.2 below.

Labeled deoxynucleotides such as that shown in Formula IV.2 findparticular application as reagents for labeling polymerase extensionproducts, e.g., in the polymerase chain reaction or nick-translation.

1.5.2. Polynucleotide Reagents

Yet another preferred class of reagents of the present inventioncomprise polynucleotides labeled with the dibenzorhodamine dyes of theinvention. Such labeled polynucleotides are useful in a number ofimportant contexts including as DNA sequencing primers, PCR primers,oligonucleotide hybridization probes, oligonucleotide ligation probes,and the like.

In one preferred embodiment, the labeled polynucleotides of the presentinvention include multiple dyes located such that fluorescence energytransfer takes place between a donor dye and an acceptor dye. Suchmulti-dye energy-transfer polynucleotides find application asspectrally-tunable sequencing primers, e.g., Ju et al., Proc. Natl.Acad. Sci. USA 92: 4347-4351 (1995), and as hybridization probes, e.g.,Lee et al. Nucleic Acids Research, 21: 3761-3766 (1993).

Labeled polynucleotides may be synthesized either enzymatically, e.g.,using a DNA polymerase or ligase, e.g., Stryer, Biochemistry, Chapter24, W.H. Freeman and Company (1981), or by chemical synthesis, e.g., bythe phosphoramidite method, the phosphite-triester method, and the like,e.g., Gait, Oligonucleotide Synthesis, IRL Press (1990). Labels may beintroduced during enzymatic synthesis utilizing labeled nucleotidetriphosphate monomers as described above, or introduced during chemicalsynthesis using labeled non-nucleotide or nucleotide phosphoramidites asdescribed above, or may be introduced subsequent to synthesis.

Generally, if the labeled polynucleotide is made using enzymaticsynthesis, the following procedure may be used. A template DNA isdenatured and an oligonucleotide primer is annealed to the template DNA.A mixture of deoxynucleotide triphosphates is added to the mixtureincluding dGTP, dATP, dCTP, and dTTP where at least a fraction of thedeoxynucleotides is labeled with a dye compound of the invention asdescribed above. Next, a polymerase enzyme is added under conditionswhere the polymerase enzyme is active. A labeled polynucleotide isformed by the incorporation of the labeled deoxynucleotides duringpolymerase-mediated strand synthesis. In an alternative enzymaticsynthesis method, two primers are used instead of one, one primercomplementary to the + strand and the other complementary to the −strand of the target, the polymerase is a thermostable polymerase, andthe reaction temperature is cycled between a denaturation temperatureand an extension temperature, thereby exponentially synthesizing alabeled complement to the target sequence by PCR, e.g., PCR Protocols,Innis et al. eds., Academic Press (1990).

Labeled polynucleotides may be chemically synthesized using thephosphoramidite method. Detailed descriptions of the chemistry used toform polynucleotides by the phosphoramidite method are providedelsewhere, e.g., Caruthers et al., U.S. Pat. Nos. 4,458,066 and4,415,732; Caruthers et al., Genetic Engineering, 4: 1-17 (1982); UsersManual Model 392 and 394 Polynucleotide Synthesizers, pages 6-1 through6-22, Applied Biosystems, Part No. 901237 (1991).

The phosphoramidite method of polynucleotide synthesis is the preferredmethod because of its efficient and rapid coupling and the stability ofthe starting materials. The synthesis is performed with the growingpolynucleotide chain attached to a solid support, so that excessreagents, which are in the liquid phase, can be easily removed byfiltration, thereby eliminating the need for purification steps betweensynthesis cycles.

The following briefly describes the steps of a typical polynucleotidesynthesis cycle using the phosphoramidite method. First, a solid supportincluding a protected nucleotide monomer is treated with acid, e.g.,trichloroacetic acid, to remove a 5′-hydroxyl protecting group, freeingthe hydroxyl for a subsequent coupling reaction. An activatedintermediate is then formed by simultaneously adding a protectedphosphoramidite nucleoside monomer and a weak acid, e.g., tetrazole, tothe reaction. The weak acid protonates the nitrogen of thephosphoramidite forming a reactive intermediate. Nucleoside addition iscomplete within 30 s. Next, a capping step is performed which terminatesany polynucleotide chains that did not undergo nucleoside addition.Capping is preferably done with acetic anhydride and 1-methylimidazole.The internucleotide linkage is then converted from the phosphite to themore stable phosphotriester by oxidation using iodine as the preferredoxidizing agent and water as the oxygen donor. After oxidation, thehydroxyl protecting group is removed with a protic acid, e.g.,trichloroacetic acid or dichloroacetic acid, and the cycle is repeateduntil chain elongation is complete. After synthesis, the polynucleotidechain is cleaved from the support using a base, e.g., ammonium hydroxideor t-butyl amine. The cleavage reaction also removes any phosphateprotecting groups, e.g., cyanoethyl. Finally, the protecting groups onthe exocyclic amines of the bases and the hydroxyl protecting groups onthe dyes are removed by treating the polynucleotide solution in base atan elevated temperature, e.g., 55° C.

Any of the phosphoramidite nucleoside monomers may be dye-labeledphosphoramidites as described above. If the 5′-terminal position of thenucleotide is labeled, a labeled non-nucleotidic phosphoramidite of theinvention may be used during the final condensation step. If an internalposition of the oligonucleotide is labeled, a labeled nucleotidicphosphoramidite of the invention may be used during any of thecondensation steps.

Subsequent to synthesis, the polynucleotide may be labeled at a numberof positions including the 5′-terminus, e.g., Oligonucleotides andAnalogs, Eckstein ed., Chapter 8, IRL Press (1991) and Orgel et al.,Nucleic Acids Research 11(18): 6513 (1983); U.S. Pat. No. 5,118,800; thephosphodiester backbone, e.g., ibid., Chapter 9; or at the 3′-terminus,e.g., Nelson, Nucleic Acids Research 20(23): 6253-6259, and U.S. Pat.Nos. 5,401,837 and 5,141,813. For a through review of oligonucleotidelabeling procedures see R. Haugland in Excited States of Biopolymers,Steiner ed., Plenum Press, NY (1983).

In one preferred post-synthesis chemical labeling method anoligonucleotide is labeled as follows. A dye including a carboxy linkinggroup is converted to the N-hydroxysuccinimide ester by reacting withapproximately 1 equivalent of 1,3-dicyclohexylcarbodiimide andapproximately 3 equivalents of N-hydroxysuccinimide in dry ethyl acetatefor 3 hours at room temperature. The reaction mixture is washed with 5%HCl, dried over magnesium sulfate, filtered, and concentrated to a solidwhich is resuspended in DMSO. The DMSO dye stock is then added in excess(10-20×) to an aminohexyl derivatized oligonucleotide in 0.25 Mbicarbonate/carbonate buffer at pH 9.4 and allowed to react for 6 hours,e.g., U.S. Pat. No. 4,757,141. The dye labeled oligonucleotide isseparated from unreacted dye by passage through a size-exclusionchromatography column eluting with buffer, e.g., 0.1 molar triethylamineacetate (TEAA). The fraction containing the crude labeledoligonucleotide is further purified by reverse phase HPLC employinggradient elution.

1.6. METHODS UTILIZING THE DIBENZORHODAMINE DYES

The dyes and reagents of the present invention are well suited to anymethod utilizing fluorescent detection, particularly methods requiringthe simultaneous detection of multiple spatially-overlapping analytes.Dyes and reagents of the invention are particularly well suited foridentifying classes of polynucleotides that have been subjected to abiochemical separation procedure, such as electrophoresis, or that havebeen distributed among locations in a spatially-addressablehybridization array.

In a preferred category of methods referred to herein as “fragmentanalysis” or “genetic analysis” methods, labeled polynucleotidefragments are generated through template-directed enzymatic synthesisusing labeled primers or nucleotides, e.g., by ligation orpolymerase-directed primer extension; the fragments are subjected to asize-dependent separation process, e.g., electrophoresis orchromatography; and, the separated fragments are detected subsequent tothe separation, e.g., by laser-induced fluorescence. In a particularlypreferred embodiment, multiple classes of polynucleotides are separatedsimultaneously and the different classes are distinguished by spectrallyresolvable labels.

One such fragment analysis method known as amplified fragment lengthpolymorphism detection (AmpFLP) is based on amplified fragment lengthpolymorphisms, i.e., restriction fragment length polymorphisms that areamplified by PCR. These amplified fragments of varying size serve aslinked markers for following mutant genes through families. The closerthe amplified fragment is to the mutant gene on the chromosome, thehigher the linkage correlation. Because genes for many genetic disordershave not been identified, these linkage markers serve to help evaluatedisease risk or paternity. In the AmpFLPs technique, the polynucleotidesmay be labeled by using a labeled polynucleotide PCR primer, or byutilizing labeled nucleotide triphosphates in the PCR.

In another such fragment analysis method known as nick translation, areaction is used to replace unlabeled nucleoside triphosphates in adouble-stranded DNA molecule with labeled ones. Free 3′-hydroxyl groupsare created within the unlabeled DNA by “nicks” caused bydeoxyribonuclease I (DNAase I) treatment. DNA polymerase I thencatalyzes the addition of a labeled nucleotide to the 3′-hydroxylterminus of the nick. At the same time, the 5′ to 3′-exonucleaseactivity of this enzyme eliminates the nucleotide unit from the5′-phosphoryl terminus of the nick. A new nucleotide with a free 3′-OHgroup is incorporated at the position of the original excisednucleotide, and the nick is shifted along by one nucleotide unit in the3′ direction. This 3′ shift will result in the sequential addition ofnew labeled nucleotides to the DNA with the removal of existingunlabeled nucleotides. The nick-translated polynucleotide is thenanalyzed using a separation process, e.g., electrophoresis.

Another exemplary fragment analysis method is based on variable numberof tandem repeats, or VNTRs. VNTRs are regions of double-stranded DNAthat contain adjacent multiple copies of a particular sequence, with thenumber of repeating units being variable. Examples of VNTR loci arepYNZ22, pMCT118, and Apo B. A subset of VNTR methods are those methodsbased on the detection of microsatellite repeats, or short tandemrepeats (STRs), i.e., tandem repeats of DNA characterized by a short(2-4 bases) repeated sequence. One of the most abundant interspersedrepetitive DNA families in humans is the (dC-dA)n-(dG-dT)n dinucleotiderepeat family (also called the (CA)n dinucleotide repeat family). Thereare thought to be as many as 50,000 to 100,000 (CA)n repeat regions inthe human genome, typically with 15-30 repeats per block. Many of theserepeat regions are polymorphic in length and can therefore serve asuseful genetic markers. Preferably, in VNTR or STR methods, label isintroduced into the polynucleotide fragments by using a dye-labeled PCRprimer.

In a particularly preferred fragment analysis method, classes identifiedin accordance with the invention are defined in terms of terminalnucleotides so that a correspondence is established between the fourpossible terminal bases and the members of a set of spectrallyresolvable dyes. Such sets are readily assembled from the dyes of theinvention by measuring emission and absorption bandwidths withcommercially available spectrophotometers. More preferably, the classesarise in the context of the chemical or chain termination methods of DNAsequencing, and most preferably the classes arise in the context of thechain termination methods, i.e., dideoxy DNA sequencing, or Sanger-typesequencing.

Sanger-type sequencing involves the synthesis of a DNA strand by a DNApolymerase in vitro using a single-stranded or double-stranded DNAtemplate whose sequence is to be determined. Synthesis is initiated at adefined site based on where an oligonucleotide primer anneals to thetemplate. The synthesis reaction is terminated by incorporation of anucleotide analog that will not support continued DNA elongation.Exemplary chain-terminating nucleotide analogs include the2′,3′-dideoxynucleoside 5′-triphosphates (ddNTPs) which lack the 3′-OHgroup necessary for 3′ to 5′ DNA chain elongation. When properproportions of dNTPs (2′-deoxynucleoside 5′-triphosphates) and one ofthe four ddNTPs are used, enzyme-catalyzed polymerization will beterminated in a fraction of the population of chains at each site wherethe ddNTP is incorporated. If labeled primers or labeled ddNTPs are usedfor each reaction, the sequence information can be detected byfluorescence after separation by high-resolution electrophoresis. In thechain termination method, dyes of the invention can be attached toeither sequencing primers or dideoxynucleotides. Dyes can be linked to acomplementary functionality on the 5′-end of the primer, e.g. followingthe teaching in Fung et al, U.S. Pat. No. 4,757,141; on the base of aprimer; or on the base of a dideoxynucleotide, e.g. via the alkynylaminolinking groups disclosed by Hobbs et al, supra.

In each of the above fragment analysis methods labeled polynucleotidesare preferably separated by electrophoretic procedures, e.g. Gould andMatthews, cited above; Rickwood and Hames, Eds., Gel Electrophoresis ofNucleic Acids: A Practical Approach, IRL Press Limited, London, 1981;Osterman, Methods of Protein and Nucleic Acid Research, Vol. 1Springer-Verlag, Berlin, 1984; or U.S. Pat. Nos. 5,374,527, 5,624,800and/or 5,552,028. Preferably the type of electrophoretic matrix iscrosslinked or uncrosslinked polyacrylamide having a concentration(weight to volume) of between about 2-20 weight percent. Morepreferably, the polyacrylamide concentration is between about 4-8percent. Preferably in the context of DNA sequencing in particular, theelectrophoresis matrix includes a denaturing agent, e.g., urea,formamide, and the like. Detailed procedures for constructing suchmatrices are given by Maniatis et al., “Fractionation of Low MolecularWeight DNA and RNA in Polyacrylamide Gels Containing 98% Formamide or 7M Urea,” in Methods in Enzymology, 65: 299-305 (1980); Maniatis et al.,“Chain Length Determination of Small Double- and Single-Stranded DNAMolecules by Polyacrylamide Gel Electrophoresis,” Biochemistry, 14:3787-3794 (1975); Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, New York, pgs. 179-185 (1982);and ABI PRISM™ 377 DNA Sequencer User's Manual, Rev. A, January 1995,Chapter 2 (p/n 903433, The Perkin-Elmer Corporation, Foster City,Calif.). The optimal electrophoresis conditions, e.g., polymerconcentration, pH, temperature, concentration of denaturing agent,employed in a particular separation depends on many factors, includingthe size range of the nucleic acids to be separated, their basecompositions, whether they are single stranded or double stranded, andthe nature of the classes for which information is sought byelectrophoresis. Accordingly application of the invention may requirestandard preliminary testing to optimize conditions for particularseparations.

Subsequent to electrophoretic separation, the dye-polynucleotideconjugates are detected by measuring the fluorescence emission from thedye labeled polynucleotides. To perform such detection, the labeledpolynucleotides are illuminated by standard means, e.g. high intensitymercury vapor lamps, lasers, or the like. Preferably the illuminationmeans is a laser having an illumination beam at a wavelength above about600 nm. More preferably, the dye-polynucleotides are illuminated bylaser light generated by a He—Ne gas laser or a solid-state diode laser.The fluorescence is then detected by a light-sensitive detector, e.g., aphotomultiplier tube, a charged coupled device, or the like. Exemplaryelectrophoresis detection systems are described elsewhere, e.g., U.S.Pat. Nos. 5,543,026; 5,274,240; 4,879,012; 5,091,652 and 4,811,218.

1.7. EXAMPLES

The invention will be further clarified by a consideration of thefollowing examples, which are intended to be purely exemplary of theinvention and not to in any way limit its scope.

1.8. MATERIALS AND METHODS

All chemicals were purchased from Aldrich Chemical Company unlessotherwise noted. Martius yellow was purchased from Fluka. Acetone wasdried over CaSO₄ and distilled. Dichloromethane (CH₂Cl₂) andnitrobenzene were dried over CaH₂ and distilled. Tetrahydrofuran (THF)was dried over lithium aluminum hydride (LAH) and distilled as needed.Triethylamine (Et₃N) was dried over sodium and distilled. DMSO (99.9%)and N,N-diisopropylethylamine (99.5%) were dried and stored overactivated molecular sieves. Silica gel (220-400 mesh) from VWR was usedfor normal phase flash chromatography. Reverse phase chromatography wasperformed on octadecyl functionalized silica gel from Aldrich.Preparative thin layer chromatography (PTLC) was performed on 1 and 2 mmpre-made silica gel plates from EM science (VWR). TLC was performed onaluminum back silica gel 60 plates from EM science (VWR). Developedspots were visualized with both long and short wavelength UVirradiation.

Absorption spectroscopy was performed on a Hewlett Packard model 8451AUV/Vis diode array spectrophotometer. Fluorescence measurements weremade on a Perkin-Elmer LS-50B luminescence spectrophotometer. NMRspectra were determined on a Varian 300 MHz NMR referenced relative to asolvent peak at 7.26 ppm in CD₃Cl or 3.31 ppm in CD₃OD. HPLCpurification of oligomer labeled dye fragments was performed on aPerkin-Elmer Series 200 pump, employing a reverse phase C-18 column,with both UV and fluorescence emission detection. Fluorescence detectionwas performed by a Perkin-Elmer LC 240 fluorescence detector equippedwith a red sensitive PMT, and UV detection was performed with a Model LC295 UV/Vis detector. Pump and detectors were all interfaced with aPerkin-Elmer Model 1022 computer run in two-channel mode. Buffers weremade up fresh from the following concentrated stock: 10×TBE (0.89 Mtris-(hydroxymethyl)aminomethane, 0.89 M borate, 0.02 Methylenediaminetetraacetic acid disodium salt), and 0.1 M TEAA(triethylamine acetate).

All reactions were run in an oven-dried round bottom flask, under argonatmosphere, and capped with a rubber septum. Anhydrous solvents weremanipulated under an argon atmosphere with oven-dried syringes. As usedherein, the term “aqueous workup” refers to a purification methodcomprising the following steps: (i) adding a reaction mixture to asaturated aqueous NH₄Cl solution, a 5% HCl solution, or a saturatedNa₂S₂O₃ solution, (ii) extracting the solution three times with anorganic solvent, e.g., EtOAc, or CH₂Cl₂, (iii) washing the combinedorganic layer once with saturated NaCl, (iv) drying the solution withNa₂SO₄, (v) filtering the drying agent, and (vi) removing the solvent invacuo. 3-Methoxy-1-hydroxynapthalene 1 was synthesized from1,3-dihydroxynapthalene by the method of K. H. Bell and L. F. McCaffery(Aust. J. Chem. 46: 731 (1993)). 1-Amino-3-methoxynapthalene 18 wassynthesized according the procedure of G. T. Morgan and E. D. Evans (J.Chem. Soc. 115: 1126 (1919)).

Example 1 Synthesis of 1-Diethylamino-3-Hydroxynapthalene 4 (FIG. 1)

3-methoxy-1-hydroxy napthalene 1 (1 gm) was suspended in dry CH₂Cl₂ (30mL). Dry triethylamine (1.2 equivalents) was added and the reaction wascooled to −5° C. Trifluoromethanesulfonic anhydride (1.1 equivalents)suspended in CH₂Cl₂ (15 mL) was added dropwise with vigorous stirringover a period of 2 hours. The reaction was allowed to come to roomtemperature and subjected to aqueous work up using 5% HCl and CH₂Cl₂.The resulting crude 3-methoxynapthalene-1-triflate 2 was purified bynormal phase flash chromatography employing an EtOAc/Hexane (1:10)mobile phase.

The purified 3-methoxynapthalene-1-triflate 2 was converted to the1-diethylamino-3-methoxynapthalene 3 using the palladium-catalyzedtriflate/amine coupling procedure of Wolfe as follows (J. P. Wolfe andS. L. Buchwald, JOC, 61: 1133 (1996)). The3-methoxy-napthalene-1-triflate 2 (1 gram) was suspended in 100 mL ofdry toluene with 0.015 equivalents of(S)-(−)-2,2′-bis(diphenylphosphino)-1,1′-binapthyl (BINAP), 0.005equivalents of tris(dibenzylideneacetone)dipalladium (Pd₂(dba)₃), and 3equivalents of dry diethyl amine. The reaction was purged with argon,and 3.3 equivalents of solid sodium t-butoxide was added with stirring.The reaction was then heated, and stirred for 16 hours at 80° C. in anoil bath. The reaction was allowed to come to room temperature andsubjected to aqueous work up using 5% HCl and CH₂Cl₂ resulting in acrude 1-diethylamino-3-methoxynapthalene 3, which was purified by normalphase flash chromatography employing EtOAc:hexane (1:49) as the mobilephase (¹HNMR: CD₃Cl d 8.20 (broad d, 1H, J=9 Hz), 7.72 (broad d, 1H,J=7.8 Hz), 7.43 (dt, 1H, J=7.2, 1.2 Hz), 7.34 (dt, 1H, J=7.7, 1.2 Hz),6.88 (d, 1H, J=2.4 Hz),), 6.82 (d, 1H, J=2.4 Hz), 3.93 (s, 3H), 3.21 (q,4H, J=7.2 Hz), 1.08 (t, 6H, J=7.2 Hz)).

Next, the methyl group of the 1-diethyl-amino-3-methoxy-napthalene 3 wasremoved by boron tribromide deprotection as follows. The1-amino-3-methoxy-napthalene (100 mg) was suspended in dry CH₂Cl₂ (5 mL)and the mixture was cooled to −70° C. in a dry ice/acetone bath. Borontribromide (10 equivalents) was added dropwise and the reaction wasstirred for 30 minutes, then placed in a refrigerator (0° C.) overnight.The reaction was quenched at −70° C. by careful addition of MeOH (10mL). Solid NaHCO₃ (30 equivalents) was added and the reaction was warmedto room temperature, then briefly heated to reflux. The mixture wascooled and filtered, the filtrate was acidified with AcOH, and thesolvent was removed in vacuo to give the crude1-diethylamino-3-hydroxynapthalene 4, which was purified by normal phaseflash chromatography employing EtOAc:hexane (1:4) as the mobile phase.

Example 2 Synthesis of N-Phenyl-3,3-Dimethyl-Hydroxy-Benzoindoline 9(FIG. 1)

The 3-methoxynapthalene-1-triflate 2 was derivatized with anilineaccording to the palladium catalyzed triflate/amine coupling reactiondescribed above in Example 1 to give the 1-anilino-3-methoxynapthalene5.

The 1-anilino-3-methoxynapthalene 5 was acetylated by an amino groupacetylation procedure as follows. The 1-amino-3-methoxynapthalene 5 (500mg) and 1.2 equivalents of dry Et₃N were suspended in 10 mL of dryCH₂Cl₂ and cooled to −5° C. using an ice/NaCl bath. 1.1 equivalent of2-bromo-2-methylpropionylchloride was added dropwise and the reactionwas stirred for 1 hour at −5° C. and stirred at room temperature for anadditional 1 hour. The reaction was allowed to come to room temperatureand subjected to aqueous work up using 5% HCl and EtOAc resulting in thecrude intermediate 1-(bromoalkyl)amido-3-methoxy-napthalene 6, which waspurified by normal phase flash chromatography employing EtOAc:hexane(1:9) as the mobile phase.

The 1-(bromoalkyl)amido-3-methoxy-napthalene 6 was cyclized using anAlCl₃ catalyzed Friedel-Crafts cyclization procedure as follows. 1 to 3equivalents of AlCl₃ in nitrobenzene was added to the1-(bromoalkyl)amido-3-hydroxy-napthalene 6. The reaction was heated to130° C. and reacted for 1 hour. Aqueous work-up using NH₄Cl and EtOAcgave the crude N-phenyl-benzoindolinone intermediate 7, which waspurified by normal phase flash chromatography employing EtOAc:hexane(1:4) as the mobile phase. The amide carbonyl group of theN-phenyl-benzoindoline intermediate 7 was then reduced with LAH to givecompound 8 (¹HNMR: CD₃Cl d 7.71 (d, 1H, J=7.8 Hz), 7.32 (m, 2H), 7.24(m, 2H), 7.07 (bt, 1H, J=6.6 Hz), 6.96 (m, 3H), 6.84 (s, 1H), 3.97 (s,3H), 3.92 (s, 2H), 1.44 (s, 6H).

Methoxy group deprotection of compound 8 was effected using the borontribromide deprotection procedure described in Example 1, resulting inthe N-phenyl-3,3-dimethyl-hydroxy-benzoindoline 9.

Example 3 Synthesis of N-Methyl-5-Hydroxy-(Tetrahydro)benzoquinoline 15(FIG. 2)

Compound 10 was synthesized by condensation of methoxy-napthaldehyde andmalonic acid employing piperidine catalysis in pyridine. Compound 10 wasreduced with hydrogen over 10% Pd/carbon, followed by LAH reduction, andreacted as outlined for the synthesis of compound 2 above withtrifluoromethanesulfonic anhydride to give the triflate 11. Triflate 11was then reacted with NaN₃ (3 equiv.) in DMF at 100° C. for 6 hours.Then, the reaction was allowed to come to room temperature and subjectedto aqueous work up using pure water and EtOAc resulting in pure compound12. Compound 12 was suspended in dry CH₂Cl₂, complexed with 3 to 5equivalents of solid AlCl₃, and refluxed for 2 hours yielding compound13.

Compound 13 was alkylated with MeI according to a general amino groupalkylation procedure as follows. The 3-methoxybenzoquinoline derivative(100 mg) 13 was suspended in 5 mL of dry THF and cooled to −5° C.(ice/NaCl). 1.1 equivalents of n-butyl lithium (1 M) was added dropwise,and the reaction was stirred for 1 hour. 3 equivalents of the MeIalkylating agent was added slowly and the reaction was allowed to stirat room temperature for 2 hours. Aqueous work-up using NH₄Cl and EtOAcgave a crude alkylated 3-methoxybenzoquinoline intermediate 14.Intermediate 14 was then purified by normal phase flash chromatographyemploying EtOAc:hexane (1:19) as the mobile phase (¹HNMR: CD₃Cl d 8.1(broad d, 1H, J=8.1 Hz), 7.68 (dd, 1 H, J=8.1, 1.8 Hz), 7.34 (m, 2H),6.8 (s, 1H), 3.92 (s, 3H), 3.21 (m, 2H), 2.94 (s, 3H), 2.77 (t, 2H,J=6.6 Hz), 1.92 (m, 2H)). Subsequent methoxy group deprotection by thegeneral boron tribromide procedure described above in Example 1 resultedin the N-methyl-hydroxybenzoquinoline derivative 15.

Example 4 Synthesis of 3-(5-Hydroxybenzoquinolin-1-yl) propanesulfonicacid 17 (FIG. 2)

Compound 13 was synthesized according to the procedure outlined above inExample 3 for the synthesis of the N-methyl-hydroxybenzoquinolinederivative 15. Compound 13 was then alkylated according to the generalamino group alkylation procedure described above in Example 3, this timeusing 1,3-propane sultone as the alkylating agent rather than MeI, togive a 5-methoxybenzoquinoline-N-propanesulfonic acid intermediate 16(1HNMR: CD3OD d 7.94 (d, 1H, J=8.7 Hz), 7.65 (d, 1H, J=8.4 Hz), 7.32 (t,1H), 7.27 (t, 1H), 6.85 (s, 1H), 4.89 (s, 3H), 3.20 (m, 2H), 3.08 (bt,2H, J=6 Hz), 2.91 (m, 2H), 2.72 (t, 2H, J=6.6 Hz), 2.33 (m, 2H), 1.89(m, 2H). Subsequent methoxy group deprotection of compound 16 by thegeneral boron tribromide procedure described above in Example 1 resultedin the 3-(5-hydroxybenzoquinolin-1-yl) propanesulfonic acid 17.

Example 5 Synthesis ofN-Methyl-2,2,4-Trimethyl-5-Hydroxy-(Tetrahydro)benzoquinoline 22 (FIG.3)

Following the procedure of A. Rosowsky and E. J. Modest (J.O.C., 30:1832 (1965)), 1-amino-3-methoxynapthalene 18 (1 gm) was dissolved in dryacetone (50 mL), and 0.01 equivalent of iodine was added to thesolution. The reaction was heated and stirred for 16 hours, cooled, andthen quenched with saturated Na2S2O3. The reaction mixture was thensubjected to aqueous work up using saturated Na2S2O3 and EtOAc resultingin the crude methoxybenzoquinoline 19. The methoxybenzoquinoline 19 waspurified by flash chromatography using an EtOAc/hexane 1:9 mobile phase.Compound 19 was then alkylated with MeI according to the general aminogroup alkylation procedure described above in Example 3 to give compound20. Compound 20 was reduced with H₂ in a Parr hydrogenator at 70 psi and10% Pd/C catalysis to give aN-methyl-2,2,4-trimethyl-5-methoxybenzoquinoline intermediate 21 (¹HNMR:CD₃Cl d 8.20 (bd, 1H, J=7.5 Hz), 7.65 (bd, 1H, J=7.5 Hz), 7.33 (m, 2H),6.89 (s, 1H), 3.94 (s, 3H), 3.14 (b sextet, 1H, J=6.6 Hz), 2.80 (3, 3H),1.89 (d, 2H, J=8.7), 1.42 (d, 3H, J=6.9 Hz), 1.34 (s, 3H), 1.05 (s, 3H).Subsequent methoxy group deprotection of compound 21 by the generalboron tribromide procedure described above in Example 1 gave theN-methyl-5-hydroxy-(tetrahydro)benzoquinoline 22.

Example 6 Synthesis of N-Methyl-3,3-Dimethyl-4-Hydroxy-Benzoindoline 27(FIG. 3)

1-Amino-3-methoxynapthalene 18 was acetylated with2-bromo-2-methylpropionyl chloride according to the general amino groupacylation procedure described above in Example 2 to give compound 23.Compound 23 was cyclized by the Friedel-Crafts cyclization proceduredescribed above in Example 2 to give compound 24. Next, compound 24 wasreduced with 3 equivalents LAH in THF to give the 4-methoxybenzoindoline25. Compound 25 was alkylated using the general amino group alkylationprocedure described above in Example 3 using methyl iodide as thealkylating agent to give a N-methyl-3,3-dimethyl-4-methoxybenzoindolineintermediate 26 (¹HNMR: CD₃Cl d 8.07 (bd, 1H, J=8.4 Hz), 7.69 (bd, 1H,J=8.1 Hz), 7.33 (bt, 1H, J=7.8 Hz), 7.22 (bt, 1H, J=8.1 Hz), 6.70 (s,1H), 3.92 (s, 3H), 3.32 (s, 2H), 3.32 (s, 3H), 1.44 (s, 6H). Subsequentmethoxy group deprotection of compound 26 by the general borontribromide procedure described in Example 1 resulted in theN-methyl-3,3-dimethyl-4-hydroxy-benzoindoline 27.

Example 7 Synthesis of N-Ethyl-3,3-Dimethyl-4-Hydroxy-Benzoindoline 29(FIG. 3)

The 4-methoxybenzoindoline 25 was synthesized as described above inExample 6. Compound 25 was alkylated by the general amino groupalkylation procedure described in Example 3 employing ethyl iodide asthe alkylating agent to give theN-ethyl-3,3-dimethyl-4-methoxybenzoindoline intermediate 28 (¹HNMR:CD₃Cl d 7.90 (d, 1H, J=8.7 Hz), 7.68 (d, 1H, J=8.1 Hz), 7.32 (bt, 1H,J=7.5 Hz), 7.22 (bt, 1H, J=6.9 Hz), 6.69 (s, 1H), 3.83 (s, 3H), 3.52 (q,2H J=7.5 Hz), 3.38 (s, 2H), 1.46 (s, 6H), 1.27 (t, 3H, J=7.5 Hz).Subsequent methoxy group deprotection of compound 28 by the generalboron tribromide procedure described in Example 1 yielded theN-ethyl-3,3-dimethyl-4-hydroxy-benzoindoline 29.

Example 8 Synthesis of Selected Dibenzorhodamine Dye Compounds

General Procedure A (FIG. 5). A solid phthalic anhydride derivative 34was mixed with 1.4 equivalents of an aminohydroxy intermediate 31 and2.8 equivalents of ZnCl₂. The oven dried reaction vessel was capped witha rubber septa and purged with Argon. The solid mixture was heatedbriefly at 130° C. until melting was observed, e.g., after approximately15 minutes. 1,2-Dichlorobenzene (approximately 10 equivalents) was addedby syringe to the reaction mixture, and the heterogeneous mixture washeated to 130° C. to 170° C. for 4 hours. The crude reaction mixture wascooled, suspended in a minimal amount of MeOH: CH₂Cl₂ (1:19), loadeddirectly onto a normal phase flash chromatography column, and the crudedye was eluted with an MeOH: CH₂Cl₂ (1:19) mobile phase. When necessary,the dye was purified and separated into distinct isomers 35 and 36 byPTLC developed with MeOH: CH₂Cl₂ (1:9). The isomerically pure dye, whichmigrated as a single spot on silica TLC eluting with 1:9 MeOH:CH₂Cl₂,was identified by its UV/Visible absorption spectra and its longwavelength fluorescent excitation and emission spectra.

General Procedure B (FIG. 6). In the general procedure outlined in FIG.6, a solid phthalic anhydride derivative 34 (100 mg) was placed in around bottom flask capped with a rubber septa and purged with dry argon.Dry nitrobenzene (2 mL) was added and heated to dissolve the anhydride.The mixture was cooled to room temperature and 3 to 6 equivalents ofanhydrous AlCl₃ was added with stirring to dissolve the solid.Subsequently, 1 equivalent of a 1-amino-3-methoxynapthalene intermediate31 was added with stirring and the reaction was heated to 130° C. for 1hour. The reaction was then cooled and suspended in EtOAc. The organiclayer was washed with saturated NH₄Cl and brine, dried over Na₂SO₄,filtered, and the solvent was removed in vacuo. When necessary, theresulting ketone intermediate 37/38 was purified and separated intodistinct isomers 37 and 38 using normal phase flash chromatography using(MeOH: CH₂Cl₂, 1:19) as the mobile phase, or by recrystallization. Themethoxy group of the isomerically pure intermediate 37 or 38 was removedaccording to the general boron tribromide deprotection proceduredescribed in Example 1 to give amino-hydroxynapthalene ketone 39. Theamino-hydroxynapthalene ketone 39 (100 mg) was then reacted at 130° C.with 1 equivalent of a 1-amino-3-napthalene intermediate 32 in dry1,2-dichlorobenzene (2 mL) for 2 hours. The reaction was cooled, givingisomerically pure and asymmetrically substituted product 40 that waspurified as in General Procedure A above.

Synthesis of Dibenzorhodamine Dye 41 (FIG. 7). General procedure A wasfollowed employing dichlorotrimellitic anhydride as the phthalicanhydride derivative, i.e., compound 34 where the substituents at C-14and C-17 are Cl and the substituent at C-15 is CO₂H, and1-diethylamino-3-hydroxynapthalene 4 as the aminohydroxy intermediate31.

Synthesis of Dibenzorhodamine Dye 42 (FIG. 7). General procedure A wasfollowed employing dichlorotrimellitic anhydride as the phthalicanhydride derivative, i.e., compound 34 where the substituents at C-14and C-17 are Cl and the substituent at C-15 is CO₂H, andN-methyl-5-hydroxy-benzoquinoline 15 as the aminohydroxy intermediate31.

Synthesis of Dibenzorhodamine Dye 43 (FIG. 7). General procedure A wasfollowed employing dichlorotrimellitic anhydride as the phthalicanhydride derivative, i.e., compound 34 where the substituents at C-14and C-17 are Cl and the substituent at C-15 is CO₂H, and5-hydroxy-benzoquinoline 17 as the aminohydroxy intermediate 31.

Synthesis of Dibenzorhodamine Dye 44 (FIG. 7). General procedure A wasfollowed employing dichlorotrimellitic anhydride as the phthalicanhydride derivative, i.e., compound 34 where the substituents at C-14and C-17 are Cl and the substituent at C-15 is CO₂H, theN-methyl-2,2,4-trimethyl-5-hydroxy-benzoquinoline 22 as the aminohydroxyintermediate 31.

Synthesis of Dibenzorhodamine Dye 45 (FIG. 7). General procedure A wasfollowed employing dichlorotrimellitic anhydride as the phthalicanhydride derivative, i.e., compound 34 where the substituents at C-14and C-17 are Cl and the substituent at C-15 is CO₂H, andN-methyl-3,3-dimethyl-4-hydroxy-benzoindoline 27 as the aminohydroxyintermediate 31.

Synthesis of Dibenzorhodamine Dye 46 (FIG. 7). General procedure A wasfollowed employing tetrafluorophthalic anhydride as the phthalicanhydride derivative, i.e., compound 34 where the substituents at C-14to C-17 are F, and N-ethyl-3,3-dimethyl-4-hydroxy-benzoindoline 29 asthe aminohydroxy intermediate 31.

Synthesis of Dibenzorhodamine Dye 47 (FIG. 7). General procedure A wasfollowed employing dichlorotrimellitic anhydride as the phthalicanhydride derivative, i.e., compound 34 where the substituents at C-14and C-17 are Cl and the substituent at C-15 is CO₂H, andN-phenyl-3,3-dimethyl-4-hydroxy-benzoindoline 9 as the aminohydroxyintermediate 31.

Example 9 Spectral Properties of Selected Dibenzorhodamine Dye Compounds

The following table presents important spectral properties of severalrepresentative dibenzorhodamine dye compounds of the invention. Allspectra were recorded at room temperature, in 1×TBE buffer and 8 M urea,for the free dye having 0.05 absorption at the dye's λ_(max, abs). Dyeconcentration was approximately 10⁻⁶ M.

Absorption Emission Full Width at Dye Maximum (nm) Maximum (nm) Half Max(nm) 41 585 614 59 42 609 634 42 43 597 637 47 44 598 640 50 45 639 65031 46 639 652 33 47 632 676 66

All publications, patents, and patent applications referred to hereinare hereby incorporated by reference to the same extent as if eachindividual publication, patent or patent application was specificallyand individually indicated to be incorporated by reference.

Although only a few embodiments have been described in detail above,those having ordinary skill in the chemical arts will clearly understandthat many modifications are possible in these embodiments withoutdeparting from the teachings thereof. All such modifications areintended to be encompassed within the scope following claims.

1-105. (canceled)
 106. A method of identifying polynucleotide classescomprising the steps of: providing multiple classes of polynucleotidesdistributed among locations in an array; wherein a first class ofpolynucleotide comprises a dibenzorhodamine dye having a structure ofthe formula:

including nitrogen- and aryl-substituted forms thereof; and furtherwherein other classes of polynucleotides are labeled with dyesconfigured to be spectrally resolvable from the dibenzorhodamine dye ofthe first class of polynucleotide and from each other; illuminating thearray with an illumination beam configured to cause the dyes tofluoresce; and identifying the classes of the polynucleotides in thearray by the fluorescence spectrum of the dyes.
 107. The method of claim106 wherein the dibenzorhodamine dye comprises a first bridging groupwhich when taken together with the C-12-bonded nitrogen and the C-12 andC-13 carbons forms a first ring structure having from 4 to 7 members;and/or a second bridging group which when taken together with theC-2-bonded-nitrogen and the C-1 and C-2 carbons forms a second ringstructure having from 4 to 7 members.
 108. The method of claim 107wherein one or both of the first and second ring structures has fivemembers.
 109. The method of claim 108 wherein the five membered ringstructure includes one gem disubstituted carbon.
 110. The method ofclaim 109 wherein the gem substituents are lower alkyl.
 111. The methodof claim 110 wherein the gem substituents are methyl.
 112. The method ofclaim 108 wherein the five membered ring is not aromatic.
 113. Themethod of claim 107 wherein the first and second ring structures are thesame.
 114. The method of claim 107 wherein the first and second ringstructures are different.
 115. The method of claim 106 wherein thedibenzorhodamine dye comprising one or more nitrogen substituentsselected from the group consisting of lower alkyl, lower alkene, loweralkyne, phenyl, aromatic, electron-rich heterocycle, polycyclicaromatic, water-solubilizing group, linking group, including substitutedforms thereof.
 116. The method of claim 115 wherein the nitrogensubstituents are selected from the group consisting of lower alkyl,phenyl, and substituted forms thereof.
 117. The method of claim 115wherein the nitrogen substituents are selected from the group consistingof substituted lower alkyl and substituted phenyl, wherein thesubstituent is linking group or sulfonate.
 118. The method of claim 115wherein the linking group is isothiocyanate, sulfonyl chloride, 4, 6,dichlorotriazinyl, succinimidyl ester, maleimide, haloacetyl oriodoacetamide.
 119. The method of claim 115 wherein the linking group iscarboxylate.
 120. The method of claim 115 wherein the nitrogensubstituents are water-solubilizing group.
 121. The method of claim 115wherein the water-solubilizing group is selected from the groupconsisting of sulfonate, phosphate, quaternary amine, sulfate,polyhydroxyl, and water-soluble polymer.
 122. The method of claim 106wherein the dibenzorhodamine dye and the polynucleotide are connected bya linkage at a position on the polynucleotide selected from the8-position of a purine nucleobase, the 7- or 8-position of a deazapurinenucleobase, the 5-position of a pyrimidine nucleus, the 5′ terminus, the3′ terminus, and the phosphodiester backbone.
 123. The method of claim122 wherein the linkage is attached to the linking group substituent ofthe dibenzorhodamine dye.
 124. The method of claim 122 wherein thelinkage is an acetylenic amido or alkenic amido linkage.
 125. The methodof claim 124 wherein the linkage has a structure of one of the followingformulae:

wherein NUC is a nucleobase of the polynucleotide; D is thedibenzorhodamine dye; R₃ is H or C₁-C₈ alkyl; and X is a moiety having astructure of one of the following formulae:

wherein R₁ is H or C₁-C₈ alkyl, and n is an integer of 1 to
 5. 126. Themethod of claim 106 wherein the dibenzorhodamine dye has a structure ofthe following formula:

including aryl-substituted forms thereof; wherein R₁ and R₂ areindependently selected from lower alkyl, lower alkene, lower alkyne,phenyl, aromatic, electron-rich heterocycle, polycyclic aromatic,water-solubilizing group, linking group, including substituted formsthereof; and the dibenzorhodamine dye is covalently attached to thepolynucleotide by a linkage connected to a linking group substituent ofR₁ or R₂.